PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. Uncontrolled cell proliferation, invasion and migration of pancreatic cancer cells are the fundamental causes of death in PDAC patients. Our previous studies showed that KLF9 inhibits the proliferation, invasion and migration of pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In this study, we found that platelet-activating factor acetylhydrolase IB3 (PAFAH1B3) is highly expressed in pancreatic cancer tissues and cells. In vitro and in vivo studies showed that overexpression of PAFAH1B3 promoted the proliferation and invasion of pancreatic cancer cells, while downregulation of PAFAH1B3 inhibited these processes. We found that KLF9 expression is negatively correlated with PAFAH1B3 expression in pancreatic cancer tissues and cells. Western blotting revealed that KLF9 negatively regulates the expression of PAFAH1B3 in pancreatic cancer tissues and cells. Rescue experiments showed that overexpression of PAFAH1B3 could partially attenuate the suppression of pancreatic cancer cell proliferation, invasion and migration induced by KLF9 overexpression. Finally, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried out, and the results showed that KLF9 directly binds to the promoter of PAFAH1B3 and inhibits its transcriptional activity. In conclusion, our study indicated that KLF9 can inhibit the proliferation, invasion, migration and metastasis of pancreatic cancer cells by inhibiting PAFAH1B3.


Haematoxylin and eosin (H&E) staining
After the slices were deparaffinized and rehydrated, haematoxylin was added to stain the nuclei for 5 min, after which the sections were rinsed with tap water.Eosin was added for 1 min, after which the sections were rinsed with tap water.Then, the samples were dehydrated, cleared and sealed.The cells were observed and photographed under an inverted microscope.

Cell culture
The human normal pancreatic ductal epithelial cell line HPDE6-C7 and human pancreatic cancer cell lines (SW1990, MIA Paca-2, BxPC-3, PANC-1, CFPAC-1, and AsPC-1) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).The cells were cultured at 37 °C and 5% CO2 in a humidified atmosphere incubator.HPDE6-C7 cells were grown in a mixture of 50% RPMI 1640 (Doctor DE Biological, Wuhan, China) supplemented with 10% FBS, 100 U/mL penicillin G, 0.1 mg/mL streptomycin and 1% nonessential amino acid 100 × solution (Doctor DE Biological, Wuhan, China) and 50% keratinocyte medium (Doctor DE Biological, Wuhan, China) supplemented with 0.025% bovine pituitary extract and 2.5 mg/L epidermal growth factor (Doctor DE Biological, Wuhan, China), and human pancreatic cancer cell lines were cultured in DMEM (Doctor DE Biological, Wuhan, China) supplemented with 10% foetal bovine serum (FBS; Gibco) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin).The medium was changed every 2 days, and the growth of the cells was observed under an inverted microscope.

Plasmid construction and lentiviral transduction
Plasmids containing short hairpin RNA (shRNA) targeting PAFAH1B3 and the negative control (shNC) were constructed by Hanbio Tech (Shanghai, China).The sequence of shPAFAH1B3 was 5′-CAC CAT CAG CCA TCA TGA CAT GTA T-3′.The sequence of the shRNA negative control (shNC) sequence was 5′-TTC TCC GAA CGT GTC ACG TAA-3′.The PAFAH1B3 overexpression plasmid was synthesized according to the NM_002573.4transcript by Hanbio Tech (Shanghai, China).Then, these plasmids were packaged into lentiviruses and used to infect SW1990 and MIA Paca-2 cells.After 48 h of infection, puromycin (1.0 µg/ml) was added to continue the culture, and screening was stopped when the blank cells were completely killed.Western blotting was performed to evaluate the infection efficiency.

CCK-8 cell proliferation assay
Cell viability was determined with a CCK-8 assay.PDAC cells were seeded into 96-well plates at 4 × 10 3 cells per well and incubated for 0 h, 24 h, 48 h, or 72 h.Then, 10 µl of CCK-8 reagent (Doctor DE Biological, Wuhan, China) was added to each well.After 1 h of incubation at 37 °C in the dark, the absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad).Five replicate wells were measured for the experimental groups and control groups, and the measurements were averaged.All the experiments were repeated three separate times.

Transwell assay
The invasion and migration abilities of SW1990 and MIA Paca-2 cells were analysed with a 24-well Transwell chamber (Biyuntian Biological Technology Co., Ltd., Shanghai, China).The experimental procedures were performed as previously described 26 .In brief, Transwell chambers precoated with Matrigel (YB356234, BD Biosciences, United States) were used for the invasion assay, and chambers without Matrigel were used for the migration assay.Prior to cell collection, the Matrigel was diluted with serum-free culture medium at a ratio of www.nature.com/scientificreports/1:10.Next, 60 μl of diluted Matrigel was added to the upper chamber, which was placed in a 24-well plate at 37 °C for 2-3 h until culture.Next, the transfected cells (2 × 10 4 cells) in 200 μl of serum-free DMEM were added to the upper chamber, and 600 μl of DMEM containing 10% FBS was added to the lower chamber.After being cultured for 48 h at 37 °C, the PDAC cells in the chamber were fixed with 4% formaldehyde for 15 min at room temperature and stained with 0.1% crystal violet at room temperature for 15 min.The cells on the upper surface of the chamber were gently removed with a thin cotton swab.Cell migration or invasion was measured, and the cells were photographed under an inverted microscope.

Scratch assay
First, the pancreatic cancer cell lines SW1990 and MIA Paca-2 were plated into 6-well plates and cultured until they reached 80-90% confluence.Then, the tip of a 100 µl pipette was used to scratch the monolayer.The floating cells were washed with PBS for removal.Then, fresh medium without FBS was added, and the cells were cultured for 24 h.At 0 and 24 h after cell scratching, cell migration was observed at the same site under a microscope, and the scratch area was measured using ImageJ.

Colony formation assay
Cells were plated in 6-well plates at a density of 500 per well, and DMEM containing 10% FBS was added for culture for approximately 10-14 days.Then, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min.Subsequently, the colonies were dyed with crystal violet for 10 min and then rinsed with water to determine the number of colonies that had formed.

EdU assay
An EdU Cell Proliferation Kit with Alexa Fluor 647 (Beyotime, Shanghai, CHN) was used to evaluate cell proliferation.Pancreatic cancer cells were incubated with 5-ethynyl-20-deoxyuridine (EdU) for 4 h and subsequently processed according to the manufacturer's instructions.The experimental procedures were performed as previously described 27 .

In vivo experiments
The ethics involved in the animal experiments are described in the "Ethical approval" section.BALB/C nude female mice aged 5-6 weeks were purchased and housed in a sterile environment with controlled temperature and light.Tumorigenicity experiment: the nude mice were randomly divided into 4 groups with 6 mice in each group.The specific groups used were as follows: SW1990 cells stably transduced with NC-or PAFAH1B3-Flagcontaining lentivirus and MIA Paca-2 cells stably transduced with shNC-or shPAFAH1B3-containing lentivirus.The cells in each group were collected and resuspended in 100 µl of DMEM, with approximately 5 × 10 6 cells.The volume of the tumours was assessed every 3 days.The tumour size was measured with Vernier callipers, and the induced tumour volume was calculated as [length × width 2 ]/2.Four weeks after inoculation, all the nude mice were sacrificed by CO2 inhalation, and the subcutaneous tumours were collected and measured.The expression of Ki-67 in the tumours of each group was detected by IHC.Metastasis experiment: Nude mice were randomly divided into 2 groups with 6 mice in each group.MIA Paca-2 cells transduced with lentivirus containing PAFAH1B3-Flag or NC were resuspended in 100 µl of DMEM, with approximately 2 × 10 6 cells in each group.A liver metastasis model of pancreatic cancer was established by spleen inoculation in nude mice 28 .Then, MIA Paca-2 cell suspensions were injected into the spleens of nude mice to establish a liver metastasis model.All the nude mice were sacrificed by CO2 inhalation 4 weeks after inoculation to observe liver metastasis, and the differences between the two groups were compared.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed with an EZ-ChIP Kit (Millipore) according to the manufacturer's protocol.Briefly, SW1990 and MIA Paca-2 cells were cross-linked with 1% formaldehyde, and the reaction was terminated after 5 min by the addition of glycine.The cells were harvested with SDS lysis buffer and sheared by sonication cycles to generate DNA fragments with an average size of 200-1000 bp for qChIP.After chromatin immunoprecipitation, the chromosomes are eluted from the antibody/protein G microbeads and decrosslinked, after which the DNA is purified using a centrifuge column.Purified DNA was analysed by quantitative real-time PCR (qPCR), and the enrichment was expressed as the fold enrichment compared with that of IgG.The ChIP primers used targeted the PAFAH1B3 promoter as follows: forward primer, 5′-ACA CAC ACA CAC ACA CAC AC-3′; reverse primer, 5′-TGT GAA AAT TCG AGG CTC CC-3′.

Dual-luciferase reporter assay
HEK-293 T cells were suspended and seeded in 24-well plates.When the number of cells reached 70%-80%, a dual-luciferase reporter assay was performed to explore the binding relationship between KL9 and the PAFAH1B3 promoter.The PAFAH1B3 promoter region fragments (wt and mut) were inserted into the pGL3-basic vector, and KLF9 was inserted into the pcDNA3.1 vector (Hanbio, China).The firefly luciferase plasmids h-PAFAH1B3 (wt and mut) or pGL3-Basic (NC) were cotransfected with KLF9 eukaryotic expression vectors (containing h-KLF9 or pc-DNA3.1)and the pRL-TK Renilla luciferase vector (internal reference) into HEK-293 T cells.The specific transfection process was as follows: 100 µl of DMEM was combined with 0.8 µg of the target plasmid (PRL:0.2µg, promoter: 0.4 µg, transcription factor: 0.2 µg), and the sample was thoroughly mixed and placed at room temperature (solution A); then, 10 µl of DMEM and 2 µl of the transfection reagent LipoFiter (Hanbio, China) were thoroughly mixed (solution B); and solution A and solution B were fully mixed and incubated at www.nature.com/scientificreports/room temperature for 20 min.The medium was replaced with fresh medium before transfection, after which the transfection mixture was mixed.Then, the HEK-293 T cells were cultured at 37 °C and 5% CO2.After 48 h, the cell lysates were extracted, and firefly and Renilla luciferase activities were detected using dual-luciferase reporter assay kits (HB-DLR-100; Hanbio, China).The luminescence signal was detected using a multifunctional microplate reader (SuPerMax3100, Shanghai Flash Spectrum Biological Technology Co., Ltd., China).The relative fluorescence ratio was calculated as follows: relative fluorescence ratio = firefly luciferase intensity/ Renilla luciferase intensity.The experiment was repeated three times.The experimental groups were as follows.

Statistical analysis
SPSS 24.0 (IBM SPSS Statistics, Armonk, NY) statistical software and GraphPad Prism 8.0 (San Diego, CA, USA) were used for the statistical analysis and mapping of the data.The measurement data are presented as the means ± standard deviations, and differences between groups were compared by t tests or nonparametric tests.
One-way analysis of variance (ANOVA) was used to analyse the IHC scores.All the experiments were performed in triplicate.The chi-square test was used to evaluate the associations between PAFAH1B3 expression and clinicopathological parameters.The Kaplan-Meier test was used for survival analysis.The Spearman method was used for correlation analysis between KLF9 and PAFAH1B3.In all the analyses, *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively.

Ethical approval
For human tissues, all methods were carried out in accordance with relevant guidelines and regulations.All the experimental protocols were approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University, China.All of the human tissues used in the present study were obtained with written informed consent from all the subjects and their legal guardians.For animals, all the experimental protocols were approved by the Animal Ethics Committee of the Second Affiliated Hospital of Nanchang University, China.All methods were carried out in accordance with relevant guidelines and regulations.All methods used are reported in accordance with the ARRIVE guidelines (https:// arriv eguid elines.org).

High expression of PAFAH1B3 in PDAC tissue and pancreatic cancer cell lines
First, using the Xiantao bioinformatics online analysis tool to analyse the expression of PAFAH1B3 in 33 common human cancers, the results showed that the expression level of PAFAH1B3 was significantly greater in tumour tissues than in corresponding normal tumours in the ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, READ, SKCM, STAD, TGCT, THCA, THYM, UCEC and UCS datasets (Fig. 1A).The abbreviations and full names of the 33 cancers considered in this study are available in Supplementary Table S1.Furthermore, the mRNA expression of the PAFAH1B3 gene in 179 pancreatic cancer tissues and 171 pancreatic tissues in the TCGA and GTEx databases was analysed.The results showed that the PAFAH1B3 gene was significantly overexpressed in pancreatic cancer tissues compared with that in pancreatic tissues (Fig. 1B).These results suggested that PAFAH1B3 was highly expressed in most tumours, including pancreatic cancer, and may be an oncogene in PDAC.To confirm the above bioinformatics results above, we used IHC to verify the differential expression of PAFAH1B3 in 85 PDAC tissues and their corresponding adjacent normal pancreatic tissues.HE staining revealed typical PDAC and paracancerous pancreatic tissues (Fig. 2A).IHC staining revealed that PAFAH1B3 was mainly expressed mainly in the cytoplasm.Compared with that in paracancerous pancreatic tissues, PAFAH1B3 was highly expressed in PDAC tissues, and the level of expression increased with the increasing of cancer grade (Fig. 2B).Western blotting and qRT-PCR were used to measure the expression level of PAFAH1B3 in 10 pairs of fresh PDAC tissues and in their paired adjacent normal pancreatic tissues.The results showed that the expression levels of PAFAH1B3 mRNA (Fig. 2C) and protein (Fig. 2D) in PDAC tissues were significantly greater than those in pancreatic tissues.In addition, the expression of PAFAH1B3 mRNA (Fig. 2E) and protein (Fig. 2F) in pancreatic cancer cell lines (SW1990, MIA Paca-2, PANC-1, BxPC-3, CFPAC-1, and AsPC-1) and the human normal pancreatic ductal epithelial cell line HPDE6-C7 was measured by qRT-PCR and Western blotting.The results showed that the expression of PAFAH1B3 in pancreatic cancer cell lines was significantly greater than that in normal pancreatic cells.SW1990 and MIA Paca-2 cells ranked in the middle of all the pancreatic cancer cell lines in terms of PAFAH1B3 expression, and we selected these two cell lines for subsequent cell experiments.In conclusion, PAFAH1B3 is highly expressed in PDAC tissues and pancreatic cell lines.

Correlations between PAFAH1B3 expression and clinicopathological features and prognosis in PDAC patients
The expression of PAFAH1B3 in 85 PDAC patients was assessed according to the IHC scoring criteria: 35 patients were in the low-expression group (−~+), and 50 patients were in the high-expression group (++~+++).Next, we analysed the correlations between PAFAH1B3 expression levels and multiple clinicopathological features of PDAC patients.The results showed that the expression of PAFAH1B3 was significantly correlated with tumour size, lymph node metastasis status and TNM stage in PDAC patients (Table 1).Kaplan-Meier survival analysis revealed that the survival time of PDAC patients with high PAFAH1B3 expression was significantly shorter than   www.nature.com/scientificreports/ that of patients with low PAFAH1B3 expression (Fig. 3).In conclusion, these results suggest that high expression of PAFAH1B3 is closely related to poor prognosis in PDAC patients.

PAFAH1B3 promotes the proliferation, invasion and migration of pancreatic cancer cells
To verify the effect of PAFAH1B3 on the biological behaviour of pancreatic cancer cells, we constructed the SW1990 and MIA Paca-2 cell lines with stable PAFAH1B3 overexpression via lentiviral transduction and verified the transduction efficiency by Western blotting.After transduction of the virus containing the PAFAH1B3-Flag construct, the expression of PAFAH1B3 in the SW1990 and MIA Paca-2 cell lines significantly increased (Fig. 4A).Moreover, overexpression of PAFAH1B3 upregulated the expression of the proliferating protein PCNA in SW1990 and MIA Paca-2 cells (Fig. 4B).The effects of PAFH1B3 on the proliferation of pancreatic cancer cells were detected by CCK-8, plate colony formation and EdU assays.The results showed that overexpression of PAFAH1B3 enhanced the proliferative capacity of SW1990 and MIA Paca-2 cells (Fig. 4C-E).The invasion and migration abilities of pancreatic cancer cells overexpressing PAFAH1B3 were assessed by scratch, transwell and invasion assays.The results showed that upregulation of PAFAH1B3 expression significantly promoted the wound healing, migration and invasion of SW1990 and MIA Paca-2 cells (Fig. 5A-C).Then, we constructed SW1990 and MIA Paca-2 cell lines with stable PAFAH1B3 downregulation via lentiviral transduction and verified the transduction efficiency by Western blotting.After transduction with the sh-PAFAH1B3-containing virus, the expression of PAFAH1B3 in the SW1990 and MIA Paca-2 cell lines significantly decreased (Fig. S1A).Downregulation of PAFAH1B3 expression significantly inhibited the expression of the proliferating protein PCNA in SW1990 and MIA Paca-2 cells (Fig. S1B).The effects of PAFAH1B3 on the proliferation of pancreatic cancer cells were detected by CCK-8, plate colony formation and EdU assays.The results showed that downregulation of PAFAH1B3 expression significantly reduced the proliferative capacity of SW1990 and MIA Paca-2 cells (Fig. S1C-E).The invasion and migration abilities of pancreatic cancer cells with downregulated PAFAH1B3 expression were examined by scratch, transwell and invasion assays.The results showed that downregulation of PAFAH1B3 expression significantly suppressed the wound healing, migration and invasion of SW1990 and MIA Paca-2 cells (Fig. S2A-C).Overall, these results confirmed that PAFAH1B3 promotes the proliferation, invasion and migration of pancreatic cancer cells.

PAFAH1B3 promotes epithelial mesenchymal transformation (EMT) in pancreatic cancer cells
To determine the effect of PAFAH1B3 expression on EMT in pancreatic cancer cells, we used Western blotting to detect changes in EMT-related proteins after the upregulation or downregulation of PAFAH1B3 in SW1990 and MIA Paca-2 cells.Compared with that in the NC group, the protein expression of E-cadherin was downregulated in the PAFAH1B3-Flag group, and the protein expression of N-cadherin, vimentin, Snail and MMP2 was upregulated (Fig. 6A).In contrast, compared with those in the sh-NC group, after downregulating the expression of PAFAH1B3, the expression of the E-cadherin protein was upregulated, and the expression of the N-cadherin, vimentin, Snail and MMP2 proteins was downregulated in the sh-PAFAH1B3 group (Fig. 6B).

PAFAH1B3 promotes the tumorigenicity and liver metastasis of pancreatic cancer cells in vivo
An in vivo experiment was performed to explore the function of PAFAH1B3 in tumorigenesis and metastasis.First, the effect of PAFAH1B3 overexpression on the proliferation of SW1990 cells in vivo was examined.The results showed that the weight, volume and Ki-67 expression level of the tumours formed by SW1990 cells transduced with lentivirus containing PAFAH1B3-Flag in nude mice were significantly greater than those in the NC group (Fig. 7A-D).Second, the effect of downregulating PAFAH1B3 on the proliferation of MIA Paca-2 cells in vivo was examined.The results showed that the weight, volume and Ki-67 expression of the tumours formed by the MIA Paca-2 cells transduced with sh-PAFAH1B3-containing lentivirus in the nude mice were significantly lower than those in the sh-NC group (Fig. 7E-H).These results indicated that PAFAH1B3 could promote the proliferation of pancreatic cancer cells in vivo.Finally, the effect of PAFAH1B3 on the metastasis of pancreatic cancer cells in vivo was examined.MIAPaca-2 cells transduced with lentiviruses containing PAFAH1B3-Flag or NC were injected into the spleen to establish a liver metastasis model.There were more intrahepatic metastases in the PAFAH1B3-Flag group than in the NC group (Fig. 8A,B), and HE staining confirmed that the tumour was an intrahepatic metastasis (Fig. 8C).In conclusion, these results suggest that PAFAH1B3 can promote the proliferation, migration and metastasis of pancreatic cancer cells in vivo.

Correlation between the expression of KLF9 and PAFAH1B3 in PDAC tissues and cells
Exploring how to inhibit the expression of PAFAH1B3 in pancreatic cancer is worthwhile.Our team previously showed that KLF9 was underexpressed in pancreatic cancer and that the proliferation, invasion and metastasis of pancreatic cancer cells were inhibited 23 .Therefore, we hypothesized that KLF9 inhibits PAFAH1B3 expression in pancreatic cancer.First, the correlation between PAFAH1B3 and KLF9 mRNA expression in 178 PDAC patients in the TCGA database was analysed using the Xiantao bioinformatics tool.The results indicated that the expression levels of KLF9 and PAFAH1B3 were negatively correlated in PDAC (R = − 0.393, p < 0.001; Spearman's correlation analysis) (Fig. 9A).Furthermore, the expression of KLF9 and PAFAH1B3 in 10 pairs of fresh PDAC tissues and paracarcinoma pancreatic tissues was analysed via IHC.KLF9 expression was high, and PAFAH1B3 expression was low in para-cancerous pancreatic tissues.In cancer tissues, KLF9 expression was low, and PAFAH1B3 expression was high.In brief, these results indicated that the expression of KLF9 was low in PDAC tissues, while the expression of PAFAH1B3 was high (Fig. 9B).Finally, we examined the regulatory effect of KLF9 on PAFAH1B3 at the pancreatic cancer cell level.Our previous study showed that the KLF9 protein was relatively highly expressed in the HPDE6-C7 cell line, while relatively low expression was found in the SW1990, BxPC-3, PANC-1 and CFPAC-1 cell lines 23 .In the present study, Western blotting was used to verify that the protein expression of KLF9 in the HPDE6-C7 cell line was significantly greater than that in the SW1990, MIAPaca-2 and PANC-1 cell lines (Fig. 9C).Therefore, we used SW1990 and MIA Paca-2 cells for subsequent experiments.To determine whether KLF9 regulates PAFAH1B3 mRNA and protein expression in SW1990 and MIA Paca-2 cell lines, we used specific KLF9 siRNAs and plasmids to effectively reduce or enforce KLF9 expression, respectively.When KLF9 was overexpressed at the mRNA and protein levels, PAFAH1B3 mRNA and protein expression decreased significantly (Fig. 9D,E).Moreover, when KLF9 was knocked down at the mRNA and protein levels, the PAFAH1B3 mRNA and protein levels significantly increased (Fig. 9F,G).In conclusion, these results suggest that PAFAH1B3 expression is inhibited by KLF9 in pancreatic cancer tissues and cells.

Overexpression of PAFAH1B3 can partially reverse the inhibitory effect of KLF9 on the proliferation, invasion and migration of pancreatic cancer cells
A rescue experiment was performed to confirm that PAFAH1B3 mediated KLF9 to inhibit the proliferation, invasion and metastasis of pancreatic cancer cells.As shown in Fig. 10A, compared with that in the control vector group, the protein expression of PAFAH1B3 in the KLF9 overexpression group was significantly downregulated.However, in the rescue group, overexpression of PAFAH1B3 partially restored the inhibition of PAFAH1B3 due to overexpression of KLF9.These results suggest that PAFAH1B3 overexpression can partially reverse the inhibitory effect of KLF9 overexpression on PAFAH1B3 at the protein level.As shown in Fig. 10B-E, overexpression of  PAFAH1B3 partially restored the proliferation, wound healing, migration and invasion of SW1990 cells, which were suppressed by KLF9 overexpression.The same results were obtained for MIA Paca-2 cells (Fig. S3A-E).In conclusion, these results suggest that KLF9 inhibits the proliferation and metastasis of pancreatic cancer cells by downregulating PAFAH1B3 expression.

KLF9 directly binds to the promoter region of PAFAH1B3 and inhibits its transcriptional activity
Next, we investigated the mechanism by which PAFAH1B3 is regulated by KLF9.First, the binding site of KLF9 on the PAFAH1B3 promoter was predicted by the JASPAR database, and the results showed that the length of the binding site on KLF9 was in the range of -319 to − 187 bp from the PAFAH1B3 promoter.Then, a ChIP assay was used to determine whether KLF9 directly binds to the PAFAH1B3 promoter in vivo.The results showed that KLF9 was recruited to the PAFAH1B3 promoter in SW1990 and MIA Paca-2 cells (Fig. 11A), indicating that KLF9 can directly bind to the promoter of PAFAH1B3.Subsequently, we performed dual-luciferase reporter assays to investigate the relationship between KLF9 and PAFAH1B3.We constructed wild-type and mutant promoters of human PAFAH1B3 in the -319 to − 187 bp region (Fig. 11B).The KLF9 overexpression plasmid pcDNA3.1-KLF9and the control plasmid pcDNA3.1 were constructed, and the experiments were carried out according to the groups in the methods section.The results of dual-luciferase reporter assays showed that, compared with that in the pcDNA3.1 group, the luciferase activity of the PAFAH1B3 promoter was significantly weakened after pcDNA3.1-KLF9was cotransfected with wild-type PAFAH1B3 promoter, indicating that KLF9 inhibited the activity of the PAFAH1B3 promoter.However, after cotransfection of pcDNA3.1-KLF9and the mutant PAFAH1B3 promoter, there was no significant change in the luciferase activity of the mutant PAFAH1B3 promoter compared with that in the pcDNA3.1 group (Fig. 11C).In summary, these results indicate that KLF9 directly binds to the promoter of PAFAH1B3 and inhibits its transcriptional activity.

Discussion
PDAC is a highly aggressive type of cancer known as the "king of cancer" 29 .Due to the insidious onset of PDAC, many patients have distant metastases at the time of diagnosis, at which point the opportunity for surgery is lost 30 .Chemotherapy, immunotherapy and targeted therapy are also not effective for treating PDAC patients.Therefore, it is of great clinical value to explore the molecular mechanism of pancreatic cancer occurrence and development and identify new effective targets.As a potential oncogene, PAFAH1B3 is one of the most common lipid-metabolizing enzymes and is highly expressed in cancer as a potential oncogene.Studies have shown that PAFAH1B3 is highly expressed in breast cancer 14 , osteosarcoma 31 , hypopharyngeal squamous cell carcinoma 32 and multiple myeloma 33 and is a poor prognostic factor in tumours.Interestingly, in this study, we measured the expression of PAFAH1B3 in pancreatic cancer tissues and cells, and the results showed that the expression of PAFAH1B3 in cancer tissues and cells was significantly increased.Correlation analysis of the pathological features and prognosis of PDAC patients revealed that high expression of PAFAH1B3 was significantly correlated with lymph node metastasis, large tumour size and advanced TNM stage and was a poor prognostic factor in PDAC patients.Therefore, PAFAH1B3 may play a key role in the development of pancreatic cancer.
The proliferative activity of tumours is an important parameter for evaluating tumour characteristics.Studies have shown that inhibiting PAFAH1B3 expression can significantly inhibit the proliferation of liver cancer 34 , osteosarcoma 31 , lung adenocarcinoma 35 , and gastric cancer cells 36 .PCNA is a nuclear protein that reflects cell proliferation and is closely related to cell proliferation 37 .Taken together, our results suggested that PAFAH1B3 can upregulate PCNA expression in pancreatic cancer cells to promote their proliferation.Next, we further studied the effect of PAFAH1B3 on the proliferation of pancreatic cancer cells in vivo through subcutaneous tumour formation experiments in nude mice and measured the percentage of Ki-67-positive tumours.Ki-67 is a nuclear antigen that plays an important role in the process of cell mitosis and is considered a marker of cell proliferation 38 .Our results showed that overexpression of PAFAH1B3 significantly enhanced the tumorigenic ability of pancreatic cancer cells, and the percentage of Ki-67-positive tumours significantly increased.However, knockdown of PAFAH1B3 produced the opposite results.Therefore, in vitro and in vivo experiments showed that the expression of PAFAH1B3 can promote the proliferation of pancreatic cancer cells.
Metastasis is one of the most basic biological features of cancer.Ninety percent of cancer patients die from metastasis, and metastasis is one of the leading causes of cancer-related death 39 .Nearly eighty percent of patients with pancreatic cancer have already developed distant metastases by the time they are diagnosed, which significantly increases the mortality rate of patients with pancreatic cancer 40 .Downregulation of PAFAH1B3 significantly attenuates the invasion and migration ability of hypopharyngeal squamous cell carcinoma 32 , stomach cancer 36 and liver cancer 34 cells.In this study, we examined the effect of PAFAH1B3 expression on the invasion and migration of pancreatic cancer cells in vitro and in vivo.The results showed that the overexpression of PAFAH1B3 significantly promoted the wound healing, invasion and migration ability of pancreatic cancer cells, which were significantly enhanced after the overexpression of PAFAH1B3 in vitro.However, knockdown of PAFAH1B3 induced the opposite effects.In vivo experimental results showed that the number of liver metastases formed by pancreatic cancer cells in the PAFAH1B3 overexpression group was significantly greater than that in the control group.
EMT is a key step in cancer cell metastasis 41 .EMT plays an important role in the invasion and metastasis of pancreatic cancer 42 .EMT is accompanied by decreased expression of the E-cadherin protein 43 and increased synthesis of the N-cadherin protein, vimentin protein and MMP protein 44 .MMP-2 is involved in the degradation and destruction of type IV and V collagen in the basement membrane of cells, thus promoting the invasion and metastasis of tumour cells to distant areas 45 .Tang et al. 35 interfered with the expression of PAFAH1B3 in lung adenocarcinoma cells, resulting in the upregulation of E-cadherin protein levels and the downregulation of N-cadherin protein and Snail1 protein levels.In this study, our results showed that PAFAH1B3 negatively regulated the protein expression of E-cadherin and positively regulated the protein expression of N-cadherin, snail1, vimentin and MMP-2.
In conclusion, PAFAH1B3 promotes the proliferation, invasion and metastasis of PDAC cells.These findings suggest that inhibiting PAFAH1B3 expression may effectively inhibit the progression of pancreatic cancer.
Therefore, studying how to inhibit PAFAH1B3 expression in pancreatic cancer cells is of great clinical value.In recent years, examining the role of the KLF family in cancer progression has become a new direction in cancer research.Multiple members of the KLF family have been shown to inhibit the proliferation and metastasis of cancer 46 .For example, KLF4 inhibits the progression of liver cancer 47 , and KLF6 inhibits the proliferation and invasion of oral cancer cells 48 .KLF9 inhibits the development of glioma 49 , liver cancer 50 and PDAC 51 .Previous studies by our research group have shown that KLF9 is under expressed in PDAC tissues and pancreatic cancer cells and that overexpression of KLF9 can inhibit the proliferation, invasion and metastasis of pancreatic cancer cells 23 ; however the underlying mechanism of this KLF9 inhibition is unknown.However, whether PAFAH1B3 is involved in the mechanism by which KLF9 inhibits pancreatic cancer progression remains unclear.
Studies have shown that PAFAH1B3 is one of the most common lipid-metabolizing enzymes involved in cancer and promotes cancer progression.The regulation of lipid metabolism is among the main functions of KLFs.Therefore, we hypothesize that the mechanism by which KLF9 inhibits PDAC progression may involve the inhibition of PAFAH1B3 expression.To verify this hypothesis, we first examined the association of KLF9 www.nature.com/scientificreports/with PAFAH1B3 in pancreatic cancer tissues and cells.There was a negative correlation between KLF9 and PAFAH1B3 expression in pancreatic cancer tissues and cells.Second, rescue experiments further demonstrated that KLF9 inhibits the proliferation, invasion and migration of pancreatic cancer cells by inhibiting the expression of PAFAH1B3.Finally, we investigated the mechanism by which KLF9 inhibits PAFAH1B3 via ChIP and dualluciferase assays.These results confirmed that KLF9 directly binds to the -319 to − 187 bp site of the PAFAH1B3 promoter and inhibits its transcriptional activity.
In conclusion, we found that PAFAH1B3 is upregulated in PDAC and that high PAFAH1B3 expression is associated with the clinical progression of PDAC.We also found that KLF9 inhibits the proliferation and metastasis of pancreatic cancer cells by downregulating PAFAH1B3 expression.Through this study, we provide a new reference for the clinical diagnosis and treatment of PDAC.This study has several limitations.On the one hand, the number of clinical specimens collected in this study was small, and the data may have selection bias; therefore, additional samples need to be collected in future research.On the other hand, as PAFAH1B3 is a lipidmetabolizing enzyme and we did not study lipid metabolism-related phenotypes, the phenotype of PAFAH1B3 needs to be clarified in further studies.

Figure 1 .
Figure 1.Differential expression of PAFAH1B3.(A) PAFAH1B3 expression in pancancer tissues from the TCGA database.(B) PAFAH1B3 expression in PDAC tissues and adjacent normal tissues based on the TCGA and GTEx databases.***p < 0.001.

Figure 2 .
Figure 2. Expression of PAFAH1B3 in PDAC clinical specimens and pancreatic cancer cell lines.(A) H&E staining showing PDAC tissue and paracancerous tissue.(B) IHC showed differences in the expression of PAFAH1B3 between paracancerous tissue and PDAC tissues of different grades.(C,D) mRNA and protein levels of PAFAH1B3 in 10 pairs of PDAC tissues and matching adjacent normal tissues measured by RT-qPCR and Western blot, respectively.(E,F) mRNA and protein levels of PAFAH1B3 in a normal pancreatic cell line and six pancreatic cancer cell lines measured by RT-qPCR and Western blot, respectively.β-Actin was used as an internal control.The data represent the average of three independent experiments.N represents the adjacent normal tissues of PDAC tissues, and T represents the PDAC tissues.*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4 .
Figure 4. Overexpression of PAFAH1B3 promoted the proliferation of pancreatic cancer cells in vitro.(A) Western blotting analyses showing the expression levels of PAFAH1B3 in SW1990 and MIA Paca-2 cells stably transduced with lentivirus containing PAFAH1B3-Flag or NC.(B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and the protein expression levels of PAFAH1B3 and PCNA were detected via Western blotting.(C-E) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and proliferation was assessed using a CCK-8 assay, cell colony formation assays and EdU incorporation assays.β-Actin was used as an internal control.Scale bars 200 μm.*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5 .
Figure 5. Overexpression of PAFAH1B3 promoted the migration and invasion of SW1990 and MIA Paca-2 cells.(A) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC for 24 h, and a cell scratch wound healing assay was performed to detect the migration abilities of SW1990 and MIA Paca-2 cells.(B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and a transwell migration assay was performed to measure the migration abilities of SW1990 and MIA Paca-2 cells.(C) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and an invasion assay was performed to detect the invasion abilities of SW1990 and MIA Paca-2 cells.The data represent the average of three independent experiments.Scale bars 200 μm.*P < 0.05.

Figure 6 . 3 Figure 7 .
Figure 6.PAFAH1B3 affects the expression of EMT-related proteins in pancreatic cancer cells (A) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting.(B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing sh-PAFAH1B3 or sh-NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting.β-Actin was used as an internal control.The data represent the average of three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8 .
Figure 8. Effects of PAFAH1B3 on MIA Paca-2 cell migration and invasion in vivo.(A,B) A liver metastatic tumour model was established via injection of pancreatic cancer cell suspensions transduced with lentivirus containing sh-PAFAH1B3 or sh-NC into the mouse spleen.Representative images of liver specimens.Arrow, metastatic tumour.(C) H&E staining of liver metastatic tumours in the two groups.**P < 0.01.

Figure 9 .
Figure 9. Correlation between KLF9 and PAFAH1B3 protein expression in pancreatic cancer tissues and cells.(A) Spearman correlation analysis of KLF9 and PAFAH1B3 in pancreatic cancer.(B) Representative images of PAFAH1B3 and KLF9 staining in PDAC tissues and adjacent normal tissues shown by IHC.(C) Protein levels of KLF9 in HPDE6-C7 cells and SW1990, MIA Paca-2 and PANC-1 cells detected via Western blotting.SW1990 and MIA Paca-2 cells were transfected with pcDNA3.1-KLF9or pcDNA3.1 and subjected to RT-PCR assays (D) and Western blotting assays (E).SW1990 and MIA Paca-2 cells were transfected with siKLF9 or siNC and subjected to RT-PCR (F) and Western blotting (G).β-Actin was used as an internal control.The data represent the average of three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 10 .
Figure 10.PAFAH1B3 rescues the inhibitory effect of KLF9 on SW1990 cell proliferation and metastasis.(A) Western blotting analysis of PAFAH1B3 protein levels in SW1990 cells treated as indicated.(B) The ability of PAFAH1B3 to restore KLF9 expression to inhibit the proliferation of SW1990 cells was detected via plate colony formation experiments.(C-E) The ability of PAFAH1B3 to restore KLF9 expression and inhibit SW1990 cell migration and invasion was detected via cell scratch assays and Transwell and invasion assays.β-Actin was used as an internal control.The data represent the average of three independent experiments.Scale bars 200 μm.*p < 0.05; **p < 0.01; ***p < 0.001.

Figure 11 .
Figure 11.KLF9 targets the promoter of PAFAH1B3 and decreases its transcriptional activity.(A) Chromatin immunoprecipitation was performed on SW1990 and MIA Paca-2 cells with an anti-KLF9 antibody (lane 3/6) and normal goat IgG (lane 2/5).The region between − 319 and − 187 bp containing KLF9-binding sites in the human PAFAH1B3 gene was amplified.Input DNA was used as a positive control (lane 1/4) (top).The qPCR data were normalized to the input and are expressed as the fold enrichment (bottom).(B) Wild-type and mutant promoters of human PAFAH1B3 in the -319 to − 187 bp region were constructed.(C) Dual-luciferase assays showed that the KLF9 overexpression plasmid significantly inhibited the transcriptional activity of the wild-type PAFAH1B3 promoter compared with that in the control group.However, there was no significant difference in the transcriptional inhibitory activity of the mutant PAFAH1B3 promoter between the KLF9 overexpression plasmid group and the control group.The data represent the average of three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.

Table 1 .
Correlation between PAFAH1B3 expression and the clinicopathological features of PDAC patients.